Factor IX (FIX) is a single-chain, 55 kDa zymogen of a serine protease encoded on the X chromosome in humans that is an important component of the intrinsic pathway of the blood coagulation cascade. Deficiency of functional FIX causes hemophilia B, also known as Christmas disease. Hemophilia B is reported to occur in 1 in 100,000 male births and, when untreated, is associated with severe and chronic morbidity resulting from uncontrolled bleeding into muscles, joints, and body cavities following injury. Until recently, treatments for FIX deficiency have included administration of natural FIX prepared from plasma derived from blood donor pools. Such treatments carry attendant risks of infection with blood-borne viruses including human immunodeficiency virus (HIV) and hepatitis C virus (HCV), as well as unwanted thrombosis and embolism. More recently, a preparation of recombinant FIX (BeneFIX®, Wyeth) became commercially available.
Certain posttranslational modifications are required for normal FIX activity. Fix is expressed as a precursor polypeptide that requires posttranslational processing to yield mature FIX. In particular, the precursor polypeptide of FIX requires vitamin K-dependent gamma carboxylation of certain glutamic acid residues in the so-called gamma-carboxyglutamate (Gla) domain and cleavage of propeptide (see FIG. 1). The propeptide is an 18-amino acid residue sequence N-terminal to the Gla domain. The propeptide binds vitamin K-dependent gamma carboxylase and then, in vivo, is cleaved from the precursor polypeptide of FIX by an endogenous protease, most likely PACE (paired basic amino acid cleaving enzyme), also known as furin and PCSK3. Without the vitamin K-dependent gamma carboxylation, the Gla domain is unable to bind calcium to assume the correct conformation necessary to anchor the protein to negatively charged phospholipid surfaces, thereby rendering Factor IX nonfunctional. Inhibition of vitamin K-dependent carboxylation by vitamin K antagonists such as warfarin is a common form of anticoagulant therapy. Even if it is carboxylated, the Gla domain also depends on cleavage of the propeptide for proper function, since retained propeptide interferes with conformational changes of the Gla domain necessary for optimal binding to calcium and phospholipid. Thus required post-translational modifications of the precursor polypeptide of FIX include both gamma carboxylation of certain glutamic acid residues by vitamin K-dependent gamma carboxylase and cleavage of the FIX propeptide, most likely by PACE, to yield mature FIX.
Mature FIX must be activated by activated Factor XI to yield Factor IXa. In the intrinsic pathway, FIX associates with a complex with activated Factor VIII, Factor X, calcium, and phospholipid, wherein FIX is activated by Factor XIa, and then Factor IXa in turn activates Factor X in concert with activated Factor VIII. Alternatively, Factors IX and X can both be activated by Factor VIIa complexed with lipidated Tissue Factor, which has been generated via the extrinsic pathway. Factor Xa then participates in the final common pathway whereby prothrombin is converted to thrombin, which in turn converts fibrinogen to fibrin.
Until now, in vitro post-translational processing of the precursor polypeptide of FIX, consistent with what was known about in vivo processing, has relied on PACE to effect cleavage of FIX propeptide. PACE is a member of a family of at least a half dozen mammalian subtilisin/Kex2p-like serine proteases known as proprotein convertases (PCs). PACE was found using sequence homology to KEX2, an enzyme in the yeast Saccharomyces cerevisiae and the first to be identified as an endoprotease involved in precursor processing. Subsequently other PC family members have been identified and found to have varying degrees of sequence identity and different substrate specificities.
EP 0246709 describes partial cDNA and amino acid sequences of furin (i.e., PACE).
Complete cDNA and amino acid sequences of human furin (i.e., PACE) were published in 1990. Van den Ouweland A M et al. (1990) Nucleic Acids Res. 18:664; Erratum in: Nucleic Acids Res. 18:1332 (1990).
U.S. Pat. No. 5,460,950, issued to Barr et al., describes recombinant PACE and the coexpression of PACE with a substrate precursor polypeptide of a heterologous protein to improve expression of active, mature heterologous protein. In one embodiment the precursor polypeptide is a precursor polypeptide of FIX.
U.S. Pat. No. 5,935,815, issued to van de Ven et al., likewise describes recombinant human furin (i.e., PACE) and the coexpression of furin with a substrate precursor polypeptide of a heterologous protein to improve expression of active, mature heterologous protein. Possible substrate precursors disclosed in this patent include a precursor of Factor IX.
Other family members in the mammalian subtilisin/Kex2p-like proprotein convertase (PC) family in addition to PACE are reported to include PC1/PC3, PC2, PC4, PC5/6 (hereinafter referred to simply as PC5), PACE4, and LPC/PC7/PC8/SPC7. While these various members share certain conserved overall structural features, they differ in their tissue distribution, subcellular localization, cleavage specificities, and preferred substrates. For a review, see Nakayama K (1997) Biochem J. 327:625-35. Similar to PACE, these proprotein convertases generally include, beginning from the amino terminus, a signal peptide, a propeptide (that may be autocatalytically cleaved), a subtilisin-like catalytic domain characterized by Asp, His, Ser, and Asn/Asp residues, and a Homo B domain that is also essential for catalytic activity and characterized by an Arg-Gly-Asp (RGD) sequence. PACE, PACE4, and PC5 also include a Cys-rich domain, the function of which is unknown. In addition, PC5 has isoforms with and without a transmembrane domain; these different isoforms are known as PC5B and PC5A, respectively. Comparison between the amino acid sequence of the catalytic domain of PACE and the amino acid sequences of the catalytic domains of other members of this family of proprotein convertases reveals the following degrees of identity: 70 percent for PC4; 65 percent for PACE4 and PC5; 61 percent for PC1/PC3; 54 percent for PC2; and 51 percent for LPC/PC7/PC8/SPC7. Nakayama K (1997) Biochem J. 327:625-35.
PACE and PACE4 have been reported to have partially overlapping but distinct substrates. In particular, PACE4, in striking contrast to PACE, has been reported to be incapable of processing the precursor polypeptide of FIX. Wasley L C et al. (1993) J Biol. Chem. 268:8458-65; Rehemtulla A et al. (1993) Biochemistry. 32:11586-90.
U.S. Pat. No. 5,840,529, issued to Seidah et al., discloses nucleotide and amino acid sequences for human PC7 and the notable ability of PC7, as compared to other PC family members, to cleave HIV gp160 to gp120 and gp41.
Nucleotide and amino acid sequences of rodent PC5 were first described as PC5 by Lusson J et al. (1993) Proc Natl Acad Sci USA 90:6691-5 and as PC6 by Nakagawa T et al. (1993) J Biochem (Tokyo) 113:132-5.
U.S. Pat. No. 6,380,171, issued to Day et al., discloses nucleotide and amino acid sequences for human PC5A, the isoform without the transmembrane domain, as well as methods for reducing restenosis by using antisense nucleic acids to inhibit PC5 activity.